Is There Genetic Diversity in the ‘Leucaena Bug’ Synergistes jonesii Which May Reflect Ability to Degrade Leucaena Toxins?

Part of the Plant Sciences Commons, and the Soil Science Commons This document is available at https://uknowledge.uky.edu/igc/22/2-4/28 The 22nd International Grassland Congress (Revitalising Grasslands to Sustain Our Communities) took place in Sydney, Australia from September 15 through September 19, 2013. Proceedings Editors: David L. Michalk, Geoffrey D. Millar, Warwick B. Badgery, and Kim M. Broadfoot Publisher: New South Wales Department of Primary Industry, Kite St., Orange New South Wales, Australia


Introduction
Leucaena leucocephala, a nutritionally rich forage tree legume, contains a non-protein amino acid, mimosine, which is degraded by ruminal bacteria to toxic metabolites 3,4-DHP and 2,3-DHP resulting in goitre-like symptoms in animals, severely restricting weight gain. Raymond Jones, in the early 1980s, discovered the 'leucaena bug' in the rumen of goats in Hawaii that degraded these toxic DHP metabolites into non-toxic compounds (Jones and Lowry 1984) which was named Synergistes jonesii (Allison et al. 1992) Subsequently, a rumen inoculum containing S. jonesii was used as an 'oral drench' for cattle, kept in continuous culture (Klieve et al. 2002) and supplied to farmers to dose cattle foraging on leucaena.
Studies on Queensland herds that received this oral drench showed that up to 50% of 44 herds grazing on leucaena had apparent subclinical toxicity based on high 3,4-and 2,3-DHP excretion in urine (Dalzell et al., 2012). In another study by Graham et al. (2013), a 16S rDNA nested PCR showed that rumen digesta from 6 out of 8 properties tested had a variant DNA profile from S. jonesii ATCC 78.1 strain, which suggested a different strain of the bacterium.
It was postulated that either the continually cultured oral inoculum may have undergone genetic modification and/or that animals could harbor other DHP degrading bacteria or S. jonesii strains with differential DHP degrading potential (McSweeney et al. unpublished). The present study looks at changes in the 16S rDNA gene at the molecular level that may suggest divergence from the type strain S. jonesii 78.1 (ATCC) in Queensland cattle as well as in cattle and other ruminants, internationally. These changes can appear as discrete mutations or 'single nucleotide polymorphisms' (SNPs) and may be correlated to their ability to degrade DHP, relative to the type strain.

Materials and Methods
Rumen fluid or faeces was collected from Australian cattle in Queensland and from cattle, sheep, goats, buffalos, native cattle and yak from Indonesia, Thailand, Vietnam, China and Brazil, mainly from local farmers. Microbial DNA was extracted from these samples and amplified with a set of 16S rDNA nested PCR primers which are specific for S. jonesii. PCR products positive for S. jonesii were then aligned against full-length S. jonesii 16S rDNA sequence for identification of SNPs.

Results
The nested PCR was able to detect S. jonesii in the majority of Australian cattle tested (Table 1). Overseas ruminants (cattle, buffalos, goats, sheep and yak), whether feeding on leucaena or not, had nested PCR detectable S. jonesii 16S rDNA sequences, suggesting that the 'leucaena bug' is indigenous to many of these animals (Table 1). In general, faecal samples failed to generate PCR products for S. jonesii from either Australian or international samples.  (Table 1). Cattle from the University of Queensland, Gatton campus, had all 4 SNPs. In animals overseas, the very same SNPs (Table 1) were also distributed ranging from frequencies of 15% (for '870' in Brazilian cattle) to 100% (all 4 SNPs in Vietnam cattle and goats). Among all the international samples analysed, only Jinnan cattle, Tibetan yak and Indonesian buffalos returned 100% identity with the type strain of S. jonesii. Interestingly, these buffalos were on 100% leucaena for 0.5-1 year and had high clearance of 3,4-and 2,3-DHP (data not shown). The Jinnan cattle and Tibetan yak were naïve to dietary leucaena. Other SNPs were spread along this fragment of the 16S rDNA whose frequencies were not consistent across animals, geographical regions or loci.

Conclusions
S. jonesii appear to be indigenous to the rumen across all types of ruminants and geographical regions tested. Classical SNPs are located in base positions 268, 306, 328 & 870. Their distribution is seen across all geographical regions and animal species; however, frequencies may vary. Other, minor mutations are distributed infrequently.